Quenched Fluorophores Conjugated to Peptides Via Linkers Containing Dithio Groups

ABSTRACT

Disclosed are dithio compounds that include a quenched fluorophore and a non-fluorophore peptide linked via a dithio bond to the fluorophore. The dithio compounds may be used in methods for detecting thiol-containing compounds or dithio-containing compounds. The dithio compounds also may be used as cellular probes where the peptide portion of the compounds targets the compounds to a specific cellular location.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119(e) toU.S. Provisional Application No. 61/268,688, filed on Jun. 15, 2009, theentire contents of which are incorporated herein by reference.

BACKGROUND

The disclosed compounds generally relate to the field of dithioreagents. In particular, the disclosed compounds generally relate toquenched fluorescent dithio reagents that are linked to a peptide via alinker containing a dithio group. The compounds may be useful fordetecting thiol-containing compounds and dithio-containing compounds.

Thiols are ubiquitous in cellular biochemistry, playing important rolesin determining protein structure (as disulfide linkages) and enzymaticmechanisms (as covalent catalysts). Furthermore, the redox state in thecell is largely regulated by the thiol/disulfide status of glutathionein the cell (i.e., GSH vs GSSG). In addition, reduced glutathione (i.e.,the thiol form or GSH) also plays a control role in drug metabolism byattacking electrophilic atoms. Therefore, thiol detection andquantitation is important in cellular biochemistry, and to date has beenaccomplished most commonly by performing UV-Visible assays usingcolorimetric reagents such as Ellman's reagent. Recently, new probes andassociated methods to detect thiols have also been reported wherebyquenched fluorophores are reacted with thiols, thereby leading to afluorescence signal (e.g., Tang et al. (2007) J. Am. Chem. Soc. 129,11666; Lin et al. (2009) Chemistry 15, 5096). These methods do notinvolve use of disulfide probes, especially those tethered to peptideswhich provide lower background signal relative to other fluorescentdisulfide compositions. Furthermore, no probes or methods have beenreported to date that permit direct detection of disulfides, especiallydisulfides inside live cells.

Fluorescence detection systems (e.g., fluorescence spectroscopy) havebeen widely used to study the structure, mechanism and function ofdifferent proteins and enzymes, and especially in enzymatic activity orbinding assays. Fluorescence detection systems are useful in that theygenerally have high sensitivity and a good dynamic range for detection.In addition, many generic fluorescent reagents are available, as well ascommercially available equipment for detecting particular reagents.Fluorescence detection systems may be amenable to high throughputscreening (e.g., using any bench-top fluorescence plate reader).Potential drawbacks associated with some fluorescence reagents mayinclude photobleaching, stability, and purity of the utilizedfluorophore. Some commercially available fluorescent labeling reagentsare mixtures of isomers or have high photobleaching or causeuncontrolled labeling, which prevents them from giving reliable andreproducible results. Some also have background fluorescent signal, andothers suffer from having a lack of selectivity for the molecule orfunctional group (e.g., thiols or disulfides) being detected.

As such, fluorescent dithio reagents are desirable. In particular,fluorescent dithio reagents that are photostable, single isomers aredesirable. Further, fluorescent dithio reagents that may be used ascellular probes are desirable.

SUMMARY

Disclosed herein are dithio compounds. The dithio compounds typicallyinclude a quenched fluorophore and a non-fluorophore peptide linked viaa dithio bond to the fluorophore. The dithio compounds described hereinmay be used in methods for detecting thiol-containing compounds ordithio-containing compounds. The dithio compounds also may be used ascellular probes where the peptide portion of the compounds targets thecompounds to a specific cellular location.

The dithio compounds may have a formula F—S—S—P, where “F” includes aquenched fluorophore and “P” includes a non-fluorophore peptide. Thefluorophore typically exhibits dequenching when the dithio bond of thedithio compound is reduced (e.g., upon reaction with a thiol-containingcompound) or when the dithio compound reacts with anotherdithio-containing compound in an exchange reaction. In the compound, thefluorophore may be quenched via an interaction between the fluorophoreand a quencher (or a quenching moiety) comprising at least one of thenon-fluorophore peptide (P) and the dithio bond of the linker (S—S). Insome embodiments, the quencher is capable of increasing or decreasingthe absorption (extinction coefficient) for the fluorophore. In otherembodiments, the fluorophore is quenched by dynamic quenching. Infurther embodiments, the fluorophore is quenched by dynamic quenchingthat occurs by fluorescence resonance energy transfer (“FRET”). In evenfurther embodiments, the fluorophore is quenched by static quenching.

The dithio compounds described herein may have a formula F—X¹—S—S—X²—Pwhere X¹ and X² may be the same or different and may include C₁₋₁₈ alkylgroups, alkenyl groups, alkynyl groups, aryl groups and combinationsthereof. The fluorophore and the quencher may be present at a selecteddistance within the compounds (e.g., at a selected linear distance forquenching to occur). For example, the fluorophore and the quencher maybe present in the dithio compounds at a distance of about 6-100angstroms, preferably 15-75 angstroms, more preferably about 30-70angstroms. In some embodiments, the fluorophore and the quencher may bepresent in the dithio compounds at a distance of about 3-100 angstroms,preferably 3-75 angstroms, more preferably about 3-50 angstroms. Infurther embodiments, the fluorophore and the quencher are present in thecompound at a distance of no more than about 20 angstroms.

The dithio compounds may include any suitable fluorophore. For example,suitable fluorophores may include a fluorophore selected from a groupconsisting of fluorescein-type fluorophores, rhodamine-typefluorophores, xanthine-type fluorophores, naphthalene-type fluorophores,carbocyanine-type fluorophores, dipyrromethene boron-type fluorophores,coumarin-type fluorophores, acridine-type fluorophores, pyrene-typefluorophores, DANSYL-type fluorophores, and lanthanide chelate-typefluorophores.

The dithio compounds include a non-fluorophore peptide linked to thefluorophore via an S—S bond. Suitable non-fluorophore peptides may bealiphatic in nature, for example, lacking aromatic amino acids such asHis, Tyr, Phe, and Trp or having a low percentage of aromatic aminoacids such as His, Tyr, Phe, and Trp relative to total amino acids(e.g., having less than about 20%, 10%, 5%, 3%, or 1% aromatic aminoacids).

Suitable non-fluorophore peptides may exhibit one or more biologicalfunctions, which may include, but are not limited to, biologicaltransport or targeting activity. For example, suitable non-fluorophorepeptides may facilitate transport or retention of the dithio compoundacross a cell membrane or cell wall or into a cell organelle.

Dithio compounds that include a linked fluorophore and peptide may beprepared by any suitable method. For example, the dithio compounds maybe prepared by reacting precursors that include: (A) a first precursorthat includes a fluorophore; (B) a second precursor that includes apeptide; and (C) a dithio reagent having the formula X¹—S—S—X², where X¹and X² may be the same or different and each includes at least onereactive group capable of reacting with the first precursor and thesecond precursor. The dithio reagent may comprise diamino diphenyldisulfide and cystamine.

In suitable embodiments of the method for preparing dithio compounds,the first precursor may include a fluorophore selected from the groupconsisting of fluorescein-type fluorophores, rhodamine-typefluorophores, xanthine-type fluorophores, naphthalene-type fluorophores,carbocyanine-type fluorophores, dipyrromethene boron-type fluorophores,coumarin-type fluorophores, acridine-type fluorophores, pyrene-typefluorophores, DANSYL-type fluorophores, lanthanide chelate-typefluorophores, as well as luciferin, which exhibits luminescence afterbeing acted on by the luciferase enzyme. The second precursor mayinclude a non-fluorophore peptide. In some embodiments, the methods mayinclude reacting precursors that include: (A) a first precursor thatincludes a fluorophore; (B) a second precursor that includes anon-fluorophore peptide; and (C) a dithio reagent. The dithio reagenttypically has a formula X¹—S—S—X², where X¹ and X² may be the same ordifferent and each includes at least one reactive group capable ofreacting with the first precursor and the second precursor. In otherembodiments, the precursors for preparing the dithio compounds include:(A) a first precursor including a fluorescein-type fluorophore; (B) asecond precursor including a non-fluorophore peptide; and (C) a dithioreagent having the formula X¹—S—S—X², where X¹ and X² may be the same ordifferent and each include at least one reactive group capable ofreacting with the first precursor and the second precursor. In furtherembodiments, the precursors for preparing the dithio compounds include:(A) a first precursor including a naphthalene-type fluorophore; (B) asecond precursor including a non-fluorophore peptide; and (C) a dithioreagent having the formula X¹—S—S—X², where X¹ and X² may be the same ordifferent and each include at least one reactive group capable ofreacting with the first precursor and the second precursor. In evenfurther embodiments, the precursors for preparing the dithio compoundsinclude: (A) a first precursor including a DANSYL-type fluorophore; (B)a second precursor including a non-fluorophore peptide; and (C) a dithioreagent having the formula X¹—S—S—X², where X¹ and X² may be the same ordifferent and each include at least one reactive group capable ofreacting with the first precursor and the second precursor.

In some embodiments, the fluorophore may be derivatized to make itsfluorescence spectrum pH independent between pH 6 and pH 8. For example,the fluorophore may be halogenated and suitable fluorophores may includea halogenated fluorescein-type fluorophore and a halogenatedrhodamine-type fluorophore. In other embodiments, the fluorescein-typefluorophore is a derivative of fluorescein in which the carboxyl groupis replaced with any group that cannot cyclize (e.g., an alkyl,haloalkyl, or halo group). In other embodiments, the fluorescein-typefluorophore is a derivative of fluorescein or an analog of fluoresceinin which the carboxyl group is linked to the cyclic nitrogen atom ofpiperazine. In further embodiments, the fluorescein-type fluorophore isa derivative of fluorescein or an analog of fluorescein in which thehydroxyl groups are oxidized to ketones or replaced with alkoxy groups(e.g., methoxy or ethoxy).

In some embodiments of the methods for preparing dithio compounds, thedithio reagent may include reactive groups, (e.g., X¹ and X² each mayinclude at least one amino group) and the first precursor and the secondprecursor each may include reactive groups (e.g., at least oneamine-reactive group). Suitable reactive groups may includeamine-reactive groups and carbonyl-reactive groups. Amine-reactivegroups may include isothiocyanate groups, carboxyl groups, succinimidylester groups, and sulfonyl groups. Carbonyl-reactive groups may includeamino groups and hydrazide. Suitable dithio reagents for preparing thedithio compounds may include cystamine and diamino-diphenyl disulfide.Suitable precursors include isothiocyanate-containing fluorophores,sulfonyl-containing fluorophores, carboxyl-containing fluorophores, andthe like.

In some embodiments of the method, the first precursor and the secondprecursor each may include at least one amine-reactive group and thedithio reagent has a formula X¹—S—S—X², where X¹ has the formula—X³—NH₂; X² has the formula —X⁴—NH₂; X³ and X⁴ may be the same ordifferent and include groups independently selected from the groupsconsisting of C₁₋₁₈ alkyl groups, alkenyl groups, alkynyl groups, arylgroups and combinations thereof. In some embodiments X³ and X⁴ may bethe same or different and include aryl groups. The dithio compoundsdescribed herein (and which may be prepared by the method) may have aformula F—X³—S—S—X⁴—P, where “F” includes a fluorophore and “P” includesa peptide. X³ and X⁴ may be the same or different and may include arylgroups.

Also disclosed herein are methods for detecting thiol-containingcompounds and/or dithio-containing compounds. In some embodiments themethods include (A) reacting a reaction mixture to form at least onereaction product; and (B) detecting the at least one reaction product.Typically, the reaction mixture will include (i) the thiol-containingcompound and/or the dithio-containing compound; and (ii) a fluorescentdithio compound as described herein. For example, suitable fluorescentdithio compounds for the detection methods include dithio compoundshaving a formula F—S—S—P, in which “F” includes a fluorophore and “P”includes a non-fluorophore peptide. In the methods for detectingthiol-containing compounds and/or dithio-containing compounds asdescribed herein, detecting the at least one reaction product mayinclude observing dequenching of the fluorophore. Detecting the at leastone reaction product may also include measuring an increase or decreasein the absorbance spectrum for the fluorophore.

The methods may be used to detect any suitable thiol-containing compoundor dithio-containing compounds. For example, the methods may be used todetect thiol-containing compounds such glutathione, mycothiol,homocysteine, cysteine-containing peptides or proteins, ADPβS, GDPβS,and combinations thereof. The methods may be used to detect alteredlevels of thiols and/or dithios in cell walls or membranes, such as inbacterial cell walls or in the chorion of embryos. Such dithios mayinclude those in oxidized membrane-bound or membrane-associated proteinssuch as the E. coli Dsb system, and its eukaryotic homologs, as well aslipid transport proteins like as apoE and lipovitellin, cytokines, andC-reactive proteins (CRP). The methods may be used to detect thiolsand/or dithios quantitatively, as in clinical or biochemical assays, orqualitatively, as in a histological stain for tissue samples.

The method may be used to detect thiol-containing compounds having aformula X—S—H, where the at least one reaction product has a formulaselected from F—S—S—X, F—S—H, P—S—H, and salts thereof. Detecting the atleast one reaction product may include detecting dequenched fluorescenceor altered absorbance of the fluorophore in a reaction product having aformula selected from D-S—S—X, D-S—H, and salts thereof. The method maybe used to detect dithio-containing compounds having a formula X—S—S—X,wherein the at least one reaction product has a formula F—S—S—X (i.e., amixed disulfide) and salts thereof. Suitable thiol- anddithio-containing compounds may include polypeptides or proteins.

The methods for detecting thiol-containing compounds and/ordithio-containing compounds as described herein may be performedcontinuously or in real-time. The methods for detecting thiol-containingcompounds and/or dithio-containing compounds may be performed in vitro,in vivo, and/or in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. illustrates that the reaction of a dithio compound and G-S—Hexhibits a rapid decrease in fluorescence first, due to formation of theF—X¹—S—S-glutathione intermediate, before increasing.

FIG. 2. illustrates that the F—X¹—S—S-glutathione mixed disulfide can betrapped by reacting the fluorescent dithio probe with G-S—S-G in thedisulfide exchange reaction.

FIG. 3. illustrates that in the presence of increasing G-S—S-G anincrease in the level of the F—X¹—S—S-glutathione mixed disulfide isobserved.

FIG. 4. illustrates analogs of glutathione (G-S—H) with nucleophilicpositions blocked.

FIG. 5. illustrates the synthesis of a dithio probe comprisingpara-methyl red, cystamine disulfide, and fluorescein.

FIG. 6. illustrates a fluorescent image of an SDS PAGE gel of rabbitmuscle glyceraldehyde-3-phosphate dehydrogenase, after labeling proteinto produce FSS-protein.

FIG. 7. illustrates a fluorescent image of an SDS PAGE gel of proteinsfrom bovine lung cells, after labeling protein to produce FSS-protein(left panel). Right panel is the analogous gel, but stained usingcoomassie.

FIG. 8. illustrates images of a zebrafish embryo, using a Nikon confocalmicroscope, showing bright field image (A) and correspondingfluorescence image (B) after excitation at 490 nm. Fish was exposed tothe dithio probe shown in order to label thiol and disulfide proteins.Panel B shows that only proteins in the chorion (identified with thearrow) were labeled.

FIG. 9. illustrates a fluorescent image of an SDS PAGE gel of proteinsfrom the labeled chorion in FIG. 8, after labeling protein to produceFSS-protein (right panel). Left panel is the analogous gel, but stainedusing coomassie. The two bands identified with arrows representfluorescently tagged proteins (FSS-protein) that were extracted andidentified using LC-MS/MS (tandem mass spectrometry) in order toidentify any thiol-containing protein(s) with potential role(s) inprotecting the developing embryo from electrophiles and/or oxidativestress.

DETAILED DESCRIPTION

Disclosed herein are dithio compounds. The dithio compounds describedherein may be used in methods for detecting thiol- and otherdithio-containing compounds. For example, the dithio compounds may bereacted with thiol- or other dithio-containing compounds to detect thethiol- or other dithio-containing compounds.

As used herein, “dithio” means the chemical group —S—S—. A “dithiocompound” is a compound that includes at least one chemical group —S—S—.As used herein, “dithio” is interchangeable with “disulfide.”.

As used herein, “thiol” means the chemical group —S—H or the ionizedform of —S—H, i.e., —S—. A “thiol-containing compound” is a compoundthat includes at least one chemical group —S—H and/or —S—.

The dithio compounds described herein typically have a formula F—S—S—P,where “F” includes a fluorophore and “P” includes a non-fluorophorepeptide. Other dithio compounds discussed herein have a formula D-S—S-Aor D-S—S-Q, where “D” is a donor fluorophore, “Q” is a quencher, and “A”is an acceptor fluorophore. Related dithio compounds and methods forusing dithio compound are described in U.S. application Ser. No.11/512,485, filed on Aug. 30, 2006 and U.S. application Ser. No.11/512,485, filed on Aug. 30, 2006, which are incorporated by referenceherein in their entireties.

As used herein, a “fluorophore” is a chemical group that can be excited(e.g., by light or a chemical reaction) to emit fluorescence. Somesuitable fluorophores may be excited by light to emit phosphorescence.As used herein, a “dye” may include a fluorophore. The dithio compoundsdescribed herein may include fluorophore selected from but not limitedto: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); AnilinBlue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTS; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate;Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein;BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); BlancophorFFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503;Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FL; Bodipy FL ATP;Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate;Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1;BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; CalciumCrimson™; Calcium Green; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP-Cyan Fluorescent Protein;CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH);CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h;Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; CoelenterazineO; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTCFormazan; Cy2™; Cy3.18; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy7™; CyanGFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine;Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrotluorescein Diacetate(DCFH); DID-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydrorhodamine123 (DHR); DU (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DiIC18(7)); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP;ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide;Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FlazoOrange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; Fluor X;FM 1-43™; FM 4-46; Fura Red™; Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP(S65T); GFP red shifted(rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntrawhiteCf; JC-1; JO-JO-1; JO-PRO-1; Laurodan; LDS 751 (DNA); LDS 751 (RNA);Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X;Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-Texas Red [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH126 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-Texas Red]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); RsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange;Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (superglow GFP); SITS; SITS (Primuline); SITS (Stilbene IsothiosulphonicAcid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; SodiumGreen; Spectrum Aqua; Spectrum Green; Spectrum Orange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC Tetramethyl Rodamine Iso ThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP;YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. As used herein, a“fluorophore” may include a salt of the fluorophore. Other suitablefluorophores may fluoresce after a chemical reaction in what is calledluminescence, such as luciferin, which is a natural fluorophore becomesluminescent after the luciferase enzymatic reaction.

Fluorophores may include derivatives that have been modified tofacilitate conjugation to another reactive molecule. As such,fluorophores may include amine-reactive derivatives such asisothiocyanate derivatives and/or succinimidyl ester derivatives of thefluorophore.

The dithio compounds may include a fluorophore selected from the groupof xanthene-type fluorophores. The group of xanthene-type fluorophorestypically includes any fluorophore that includes a xanthene group havingthe formula:

Xanthene-type fluorophores include fluorescein-type fluorophores (e.g.,fluorescein and fluorescein isothiocyanate, and the like) andrhodamine-type fluorophores (e.g., rhodamine, rhodamine-B, and thelike).

The dithio compounds may include a fluorophore selected from the groupof fluorescein-type fluorophores. The group of fluorescein-typefluorophores typically includes any fluorophore that includes afluorescein group having the formula:

and derivatives and isomers thereof. Particularly useful derivativesinclude those with the carboxyl group replaced with any group thatcannot cyclize (e.g., alkyl, haloalkyl, and halo groups). Otherderivatives include those in which the carboxyl group is reacted withseparate molecule (e.g., a nitrogen atom present in a separatemolecule). For example, a derivative may be prepared by reactingfluorescein and piperazine where the carboxyl group of fluoresceinreacts with the cyclic nitrogen atom of piperazine to form an amidelinkage. The hydroxyl groups of the fluorescein molecule may be oxidizedto ketones to form derivatives. The hydroxyl groups may be replaced withalkoxy groups to form derivatives (e.g., derivatives having methoxy orethoxy groups in place of the hydroxyl groups).

Fluorescein-type fluorophores include fluorescein, fluoresceinderivatives that include a fluorescein group, and salts thereof (e.g.,5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); DCFH(Dichlorodihydrofluorescein Diacetate); Fluorescein isothiocyanate(FITC); Fluorescein Diacetate, and the like).

The dithio compounds may include a fluorophore selected from the groupof rhodamine-type fluorophores. The group of rhodamine-type fluorophorestypically includes any fluorophore that includes a rhodamine grouphaving the formula:

and isomers thereof.

Rhodamine-type fluorophores include rhodamine, rhodamine derivativesthat include a rhodamine group, and salts thereof (e.g.,5-Carboxytetramethylrhodamine (5-TAMRA); 5-ROX (carboxy-X-rhodamine);6-Carboxyrhodamine 6G; DHR (Dihydrorhodamine 123); Lissamine Rhodamine;Lissamine Rhodamine B; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD;Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; RhodamineBB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; RhodaminePhalloidine; Rhodamine Red; Sulphorhodamine B can C; Sulphorhodamine GExtra; Tetramethylrhodamine (TRITC); X-Rhodamine; XRITC, and the like).

The dithio compounds may include a fluorophore selected from the groupof the naphthalene-type fluorophores. The naphthalene-type fluorophorestypically include any fluorophore that includes a naphthalene grouphaving the formula:

Naphthalene-type fluorophores include naphthalene, IAEDANS, EDANS, andthe like. Naphthalene-type fluorophores may include pyrene.

The fluorophore may include luciferin which is luminescent afterreacting with ATP in a reaction catalyzed by luciferase. For example,the dithio compound may have a formula:

The dithio compounds described herein typically have a formula F—S—S—P,where “F” includes a fluorophore and “P” includes a non-fluorophorepeptide. As used herein, a “non-fluorophore peptide” is a peptide thatdoes not fluoresce when exposed to light (e.g., a peptide that does notfluoresce when exposed to visible light having a wavelength within arange of about 380-760 nm). In the compounds, the fluorophore typicallyexhibits dequenching when the dithio bond of the fluorophore is reduced.In the compounds, the fluorophore may be quenched via an interactionbetween the fluorophore and a quencher (or a quenching moiety)comprising at least one of the non-fluorophore peptide (P) and thedithio bond of the linker (S—S).

As used herein, the term “peptide” refers to a polymer of amino acidresidues joined by amide linkages. The term “amino acid residue,”includes but is not limited to amino acid residues contained in thegroup consisting of alanine (Ala or A), cysteine (Cys or C), asparticacid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F),glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine(Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asnor N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R),serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan(Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acidresidue” also may include amino acid residues contained in the groupconsisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine,3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino-propionic acid,allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline,4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproicacid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine,2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyricacid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine,2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline,2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid,Ornithine, and N-Ethylglycine.

Suitable peptides for the dithio compounds contemplated herein may beprimarily aliphatic in nature. For example, suitable peptides mayinclude no aromatic amino acids or a low percentage of aromatic aminoacids relative to total amino acids (e.g., where suitable peptidesinclude no more than 20%, 10%, 5%, 3%, 1% aromatic amino acids, oralternatively include at least about 80%, 90%, 95%, 97%, 99% aliphaticamino acids). The term “aromatic amino acid” includes those amino acidshaving one or more aromatic moieties such as histidine (His or H),phenylalanine (Phe or F), tryptophan (Trp or W), and tyrosine (Tyr orY). The term “aliphatic amino acid”, includes, but is not limited toamino acids in the group consisting of alanine (Ala or A), cysteine (Cysor C), aspartic acid (Asp or D), glutamic acid (Glu or E), glycine (Glyor G), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L),methionine (Met or M), asparagine (Asn or N), proline (Pro or P),glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine(Thr or T), and valine (Val or V).

In the dithio compounds, the peptide is linked to the fluorophore viathe disulfide linker. The peptide may be conjugated to the disulfidelinker via an N-terminal amino group of the peptide, via a C-terminalcarboxyl group of the peptide, or via a reactive group present in a sidechain of one of the amino acids of the peptide. For example, the peptidemay be conjugated to the disulfide linker via an amino group (e.g., of alysine residue), a carboxyl group (e.g., of an aspartic acid residue orglutamic acid residue), or thiol group (e.g., of a cysteine residue)present in a side chain of one of the amino acids.

Typically, the amide linkages of the peptides are formed from an aminogroup of the backbone of one amino acid and a carboxyl group of thebackbone of another amino acid. However, in some instances the amidelinkages may be formed from an amino group of the backbone of one aminoacid (e.g., cysteine) and a carboxyl group of a side chain of anotheramino acid (e.g., glutamate), such as in the glutathione tripeptidewhich may be present as a “non-fluorophore peptide” as contemplated inthe fluorescent dithio compound contemplated herein. For example, thepeptide of the dithio compound may comprise glutathione and the dithiocompound may have a formula:

A peptide is defined as a short polymer of amino acids, of a lengthtypically of 20 or less amino acids, and more typically of a length of12 or less amino acids (Garrett & Grisham, Biochemistry, 2^(nd) edition,1999, Brooks/Cole, 110). In some embodiments, a peptide as contemplatedherein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A polypeptide, alsoreferred to as a protein, is typically of length≧100 amino acids(Garrett & Grisham, Biochemistry, 2″ edition, 1999, Brooks/Cole, 110). Apolypeptide, as contemplated herein, may comprise, but is not limitedto, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about140, about 150, about 160, about 170, about 180, about 190, about 200;about 210, about 220, about 230, about 240, about 250, about 275, about300, about 325, about 350, about 375, about 400, about 425, about 450,about 475, about 500, about 525, about 550, about 575, about 600, about625, about 650, about 675, about 700, about 725, about 750, about 775,about 800, about 825, about 850, about 875, about 900, about 925, about950, about 975, about 1000, about 1100, about 1200, about 1300, about1400, about 1500, about 1750, about 2000, about 2250, about 2500 or moreamino acid residues. The peptides of the dithio compounds disclosedherein typically are predominantly aliphatic and include few or noaromatic amino acids (less than about 20%, 10%, 5%, 3%, or 1% aromaticamino acids.) The polypeptides detected by the methods disclosed hereintypically include one or more aromatic amino acids (and may include atleast about 1%, 3%, 5%, or 10% aromatic amino acids).

A peptide as contemplated herein may be further modified to includenon-amino acid moieties. Modifications may include but are not limitedto acylation (e.g., O-acylation (esters), N-acylation (amides),S-acylation (thioesters)), acetylation (e.g., the addition of an acetylgroup, either at the N-terminus of the protein or at lysine residues),formylation lipoylation (e.g., attachment of a lipoate, a C8 functionalgroup), myristoylation (e.g., attachment of myristate, a C14 saturatedacid), palmitoylation (e.g., attachment of palmitate, a C16 saturatedacid), alkylation (e.g., the addition of an alkyl group, such as anmethyl at a lysine or arginine residue), isoprenylation or prenylation(e.g., the addition of an isoprenoid group such as farnesol orgeranylgeraniol), amidation at C-terminus, glycosylation (e.g., theaddition of a glycosyl group to either asparagine, hydroxylysine,serine, or threonine, resulting in a glycoprotein). Distinct fromglycation, which is regarded as a nonenzymatic attachment of sugars,polysialylation (e.g., the addition of polysialic acid), glypiation(e.g., glycosylphosphatidylinositol (GPI) anchor formation,hydroxylation, iodination (e.g., of thyroid hormones), andphosphorylation (e.g., the addition of a phosphate group, usually toserine, tyrosine, threonine or histidine).

The peptides present in the dithio compounds disclosed herein mayexhibit one or more biological functions. In some embodiments, thepeptide may exhibit biological transport or targeting activity (e.g.,facilitating transport or retention of the dithio compound across a cellmembrane or cell wall or into a cell organelle such as a nucleus,endoplasmic reticulum, mitochondria, or peroxisome). Peptides thatexhibit biological transport or targeting activity include, but are notlimited to, peptides comprising signal peptide sequences for eukaryoticor prokaryotic cells, the human immunodeficiency virus “Tat” peptide(e.g., the arginine-rich RNA-binding motif (ARM)), thearginine-glycine-aspartic acid or “RGD” peptides which target integrinsand cancer cells, and the hormone ligands for cell surface receptorssuch as G-coupled protein cell surface receptors. (See, e.g., Said etal., Cell Mol Life Sci. (2010), 67(5):715-26. Epub 2009 Nov. 7; Schmidtet al., FEBS Lett. (2010) May 3, 584(9):1806-13. Epub 2009 Nov. 16; Laiet al, Traffic. (2009) Sep., 10(9):1243-56. Epub Jun. 15; Ozawa et al.,AAPS J. 2010 May 6. [Epub ahead of print]; Colette et al., J Pept Sci.(2007) Sep., 13(9):568-74; Rashid et al., (2009): “Hmrbase: a databaseof hormones and their receptors,” BMC Genomics 10(1): 307; Khar HengChoo et al.,“SPdb—a signal peptide database,” BMC Bioinformatics 2005,6:249; Cardarelli et al., Traffic (2008), 9:528-539 (describing thearginine-rich RNA-binding motif (ARM) of the Tat peptide); ErkkiRuoslahti, Annu. Rev. Cell Dev. Biol. (1996) 12:697-715; F. Sargent,Biochemical Society Transactions (2007), 35(5): 835-847; Cardarelli etal., Am. Soc. Gene Therapy (2007), 15(7):1313-1322; and Berks et al.,Molec. Microbiology (2000) 35(2), 260-274; the contents of which areincorporated by reference herein in their entireties).

Peptides may be synthesized by any technique known to those of skill inthe art, including the expression of peptides through standard molecularbiological techniques, the isolation of proteins or peptides fromnatural sources, or the chemical synthesis of proteins or peptides. Thenucleotide and peptide sequences corresponding to various genes havebeen previously disclosed, and may be found at computerized databasesknown to those of ordinary skill in the art. One such database is theNational Center for Biotechnology information's Genbank and GenPeptdatabases. The coding regions for known genes may be amplified and/orexpressed using the techniques disclosed herein or as would be know tothose of ordinary skill in the art. Peptides also can be prepared usingsynthetic organic chemistry methods, such as solid phase synthesis wherean amino acid's carboxylic acid is activated for amide bond formationwith dicyclohexyl carbodiimide (Garrett & Grisham, Biochemistry, 2^(nd)edition, 1999, Brooks/Cole, 150). Alternatively, various commercialpreparations of peptides and polypeptides are known to those of skill inthe art.

The disclosed compounds include a fluorophore and a quencher, which maycomprise at least one of the non-fluorophore peptide (P) and the dithiogroup (S—S) of the linker. In some embodiments, the fluorophore has anemission spectrum and the quencher has an absorption spectrum, such thatthe emission spectrum and absorption spectrum overlap. In particular,the emission spectrum and the absorption spectrum may overlap by about20-100%, preferably about 40-100%, more preferably about 60-100%, andeven more preferably about 70-100%. Overlap may be determined bydetermining the integral (i.e., area under the curve) for the absorbanceversus wavelength for a given range of wavelengths (e.g., λ=450-750 nm)for any selected fluorophore and any selected quencher. Quenching mayinclude dynamic quenching, static quenching, or both. Dynamic quenchingmay occur by FRET. Quenching may be relieved when the dithio group ofthe dithio compound is reduced, e.g., by reacting the dithio compoundwith a thiol-group that reduces the dithio compound. The quencher may becapable of altering the absorbance spectrum of the fluorophore. Thisalteration in absorbance may be relieved when the dithio group of thedithio compound is reduced, e.g., by reacting the dithio compound with athiol-group that reduces the dithio compound. Quenching also may berelieved when the dithio compound undergoes a disulfide exchangereaction to form a more fluorescence mixed disulfide (e.g., by reactingthe dithio compound with a disulfide on a protein), thereby forming newdisulfide bonds between the probe and the protein.

The dithio compounds may include a fluorophore and a quencher that arepresent at a selected distance within the compounds, e.g., at a distanceof about 6-100 angstroms, preferably 15-75 angstroms, more preferablyabout 30-70 angstroms. In some embodiments, the fluorophore and thequencher are present at a distance of about 3-100 angstroms, preferably3-75 angstroms, more preferably about 3-50 angstroms. The fluorophoreand the quencher may be present in the dithio compounds at a distancethat is suitable to permit FRET. In some embodiments, the fluorophoreand the quencher are present in the compound at a distance of no morethan about 20 angstroms. The fluorophore and the quencher may be presentin the dithio compounds at a distance that is suitable for staticquenching. In some embodiments, the quencher is the disulfide itself,which is only able to quench if the peptide is predominantly comprisedof aliphatic amino acids (e.g., where the peptide comprises at leastabout 80%, 90%, 95%, 97%, or 99% aliphatic amino acids, or includes noaromatic amino acids and is 100% aliphatic amino acids).

The distance between the fluorophore and the quencher may be designed byselecting a dithio linker that has a selected length. As used herein, adithio linker may have a formula —X¹—S—S—X²—, the X¹ group and the X²group may be the same or different and selected from C₁₋₁₈ alkyl groups,alkenyl groups, alkynyl groups, aryl groups and combinations thereof,optionally substituted with at least one reactive group (e.g., —NH₂ or—COOH). The length of the dithio linker may be designed by selecting asuitable X¹ group and a suitable X² group, e.g., an X¹ group and an X²group that have a suitable number of carbon atoms to provide a selectedlength for the dithio linker. Suitable linkers include cystamine orcystamine derivatives, such as compounds having the formula:

The dithio compound may have a formula F—X¹—S—S—X²—P, in which “F”includes a fluorophore and “P” includes a peptide, and at least one ofthe X¹ group and the X² group include a chemical group that is capableof influencing at least one of the emission spectrum and absorbancespectrum of the fluorophore. At least one of the X¹ group and the X²group may include a chemical group that is capable of influencing theemission spectrum of the fluorophore. At least one of the X¹ group andthe X² group may include an aryl group or an allyl group. In someembodiments, the aryl group may be selected from a phenyl group and apyridinyl group, which may be optionally substituted with at least oneof alkyl groups, haloalkyl groups, halogen groups, alkyl ester groups,ether groups, carboxyl groups, amide groups, and nitro groups.

For example, at least one of X¹ and X² may include an aryl group. Thearyl group may be substituted with an amide group. In some embodimentsat least one of X¹ and X² includes a group having a formula selectedfrom:

wherein Y¹, Y², Y³, and Y⁴ may be the same or different and are hydrogenor halide (i.e., H, F, Cl, Br, or I).

In one suitable embodiment, at least one of X¹ and X² includes a grouphaving a formula:

The dithio compound may include derivatives of cystamine ordiaminodiphenyldisulfide (herein referred to as “DAPS”). In someembodiments, the dithio compound may include derivatives ofp,p′-diaminodiphenyldisulfide, m,m′-diaminodiphenyldisulfide, ando,o′-diaminodiphenyldisulfide.

In suitable embodiments, the dithio compound has the formula:

in which R¹ includes a fluorophore group and R² includes anon-fluorophore peptide group.

The dithio compound may have a formula “FITC-DAPS-P” or “P-DAPS-FITC”,which are used interchangeably herein to refer to a dithio compound thatincludes a fluorescein-type fluorophore and a non-fluorophore peptidelinked by a diaminodiphenyldisulfide linker, (which may includep,p′-diaminodiphenyldisulfide linkers, m,m′-diaminodiphenyldisulfidelinkers, and o,o′-diaminodiphenyldisulfide linkers). “F-CYST-P” and“P-CYST-F” are used interchangeably herein to refer to a dithio compoundthat includes a fluorescein-type fluorophore and a non-fluorophorepeptide linked by a cystamine linker.

In some embodiments, the dithio compounds may include a fluorescein-typefluorophore as a fluorophore and a non-fluorophore peptide, which arepresent in the dithio compounds at a distance of about 10-60 angstroms(e.g., 40-60 angstroms). The dithio compounds may include anaphthalene-type fluorophore as a fluorophore and a non-fluorophorepeptide, which are present in the dithio compounds at a distance ofabout 10-60 angstroms (e.g., 40-60 angstroms). The dithio compounds mayinclude a DANSYL-type fluorophore as a fluorophore and a non-fluorophorepeptide, which are present in the dithio compounds at a distance ofabout 10-50 angstroms (e.g., 25-45 angstroms).

The dithio compounds may be prepared by any suitable method. Forexample, the dithio compounds may be prepared by reacting precursorsthat include: (A) a first precursor that includes a fluorophore; (B) asecond precursor that includes a peptide; and (C) a dithio reagent(i.e., a dithio linking reagent) having the formula X¹—S—S—X², where X¹and X² may be the same or different and each includes at least onereactive group capable of reacting with the first precursor and thesecond precursor. The fluorophore and the peptide may include reactivegroups as described herein. The dithio compounds that include afluorophore and a peptide (and which may be prepared by the methodsdescribed herein) may have a formula F-X³—S—S—X⁴—P where “F” includes afluorophore and “P” includes a peptide X³ and X⁴ may be the same ordifferent and may include aryl groups.

For example, the dithio compounds may be prepared by reacting a reactionmixture that includes: (a) a dithio linking agent with two or more firstreactive groups; (b) a fluorophore having at least one second reactivegroup; and (c) a peptide having a third reactive group. The second andthird reactive groups may be the same or different. In some embodiments,the fluorophore (or peptide) may be reacted with the dithio linker toform an intermediate reaction product that is at least partiallypurified and subsequently reacted with the peptide (or fluorophore,respectively). Suitable reactive groups may include nucleophilic groupsand electrophilic groups, (e.g., nucleophilic groups and electrophilicgroups capable of reacting with each other). Reactive groups may includeamino groups and amine-reactive groups (e.g., isothiocyanate groups,succinimidyl ester groups, carboxyl groups, sulfonyl groups, and thelike). Suitable dithio linking agents for preparing the dithio compoundsmay include cystamine and diaminodiphenyl disulfide.

Amine-reactive fluorophores may be derivatized by reacting with groupssuch as isothiocyanates (yielding thioureas) or succinimidyl esters(yielding carboxamides). Reactions then may be performed with dithiolinking reagents that include a reactive amine (e.g., —NH₂) and a dithiogroup. For example, the dithio linking reagent may a formulaNH₂—X¹—S—S—X²—NH₂, in which X¹ and X² may be the same or different andmay include C₁₋₁₈ (preferable C₁₋₁₈) alkyl, alkenyl, alkynyl, or aryl,which may be optionally substituted with at least one heteroatomselected from N, P, and O. To separate reaction products (e.g., F—S—S—F,F—S—S-Q and Q-S—S-Q, where F=fluorophore and Q=quencher) HPLC may beperformed. Useful reagents for synthesizing dithio compounds ascontemplated herein may include, but are not limited to, cysteine,β-mercaptoethanamine, cystamine, diamino diphenyl disulfide, andmixtures thereof.

Suitable peptides for synthesizing the dithio compounds contemplatedherein may include a cysteine residue. In some embodiments, a cysteineresidue is added to the peptide at the N-terminus or C-terminus of thepeptide. For example, the dithio compounds contemplated herein may havea formula:

When a peptide contains a thiol, such as would be the case when thepeptide contains a cysteine or homocysteine amino acid, it may bedesirable to form a disulfide of that peptide by oxidizing the thiol toa disulfide:

2 Peptide-SH=>Peptide-S—S-Peptide+2H⁺  (1)

This reaction is well known in the art, and can be facilitated, in anumber of ways, including treatment in basic conditions, or exposure toa mild oxidizing agent. For example, one can treat with iodine (I₂) orsimply air-oxidize (so O₂ is the oxidant). This reaction is described inintroductory organic chemistry textbooks, such as Organic Chemistry 5e,Brown, Foote, Iverson & Anslyn, Brooks/Cole, 2011, p. 413. One can alsooxidize the thiol to a disulfide using potassium ferricyanide, asdescribed by Hope, Murti and Vigneaud (1962) J. Biol. Chem. 237, p.1564. Another method, using silyl chloride-sulfoxide, was described byAkaji et al. (J. Am. Chem. Soc. (1992) 114, 4137-4143). A method to formdisulfides on-resin, as part of a solid phase synthesis, was describedby Galande et al. ((2005) J. Comb. Chem. 7, 174-177). A particularlysimple and effective way of forming disulfides was reported by Tam etal. ((1991) J. Am. Chem. Soc. 113, 6657-6662) using DMSO as oxidant,such as by incubating in a 20% aqueous solution.

After the disulfide is formed, one can perform a disulfide exchangereaction with a fluorophore tethered via a disulfide bind, such asF-linker-S—S-linker-F where F is a fluorophore, such as fluorescein, orone of the Alexa or Cy dyes, and “linker” could refer to the aromaticlinker, such as that in our present probe using the para-substitutedbenzene (4-thio-aniline), or the cystamine linker. This disulfideexchange reaction would proceed as follows:

Peptide-S—S-Peptide+F-linker-S—S-linker-F=>Peptide-S—S-linker-F  (2)

as a mixture of products. The desired Peptide-S—S-linker-F mixeddisulfide could then be purified, by methods such as HPLC, using areversed phase resin (ex. a C18 column), or by silica gelchromatography. The peptide used could be any peptide desired, as longas it contains one cysteine amino acid, preferable at the amino orcarboxy terminus. In some embodiments, the fluorophore is quenched mosteffectively when an aliphatic peptide of length≦20 and preferably ≦12amino acids is used. For example, if the peptide is the glutathionetripeptide, the fluorophore is completely quenched (FIG. 2). Methods forpeptide synthesis, including solid phase synthesis, are well known inthe art, companies provide peptides as contract services, and there areeven automated peptide synthesis machines to provide peptides of anydesired sequence. Especially desirable peptides for the disclosedmethods are those that signal for transport to cellular organelles, suchas the nucleus, mitochondria, peroxisomes, the golgi apparatus, and theendoplasmic reticulum. Other useful peptides are those that targetmolecules to cancer cells, such as the RGD peptides. Still other usefulpeptides are those that facilitate transport across cell walls ormembranes, such as the Tat peptides. Still other useful peptides arehormones that frequently target and bind to cell surface receptors, suchas GPCR proteins. Such peptides include, but are not limited to, growthhormone, insulin, various growth factors (ex, VEGF, TGF) and hormones,as well as neuroactive peptides.

In some embodiments of the invention, one would use purified peptide,such as that prepared in reaction (2), in cell biology studies (ex.imaging cells using confocal microscopy). In other embodiments, onemight provide the F-linker-S—S-linker-F as part of a kit, with aprocedure for the user to react their Peptide-SH (orPeptide-S—S-Peptide) of interest, in situ, to produce their ownPeptide-S—S-linker-F for cellular studies, such as confocal imaging ofthiols or disulfides in cells.

Also disclosed herein are methods for detecting thiol-containing and/ordithio-containing compounds. In some embodiments the methods include (A)reacting a reaction mixture to form at least one reaction product; and(B) detecting the at least one reaction product. Typically, the reactionmixture will include (i) the thiol-containing compound and/or thedithio-containing compound; and (ii) a fluorescent dithio compound asdescribed herein. The reaction mixture may be formed in vitro, in vivo,or in situ. The reaction mixture may be present in a cell. Thefluorescent dithio compounds may be used to detect thiol-containingcompounds and/or dithio-containing compounds in biopsy methods. Thefluorescent dithio compounds may be used to detect thiol-containingcompounds and/or dithio-containing compounds by monitoring tissue samplefluorescence levels quantitatively in a fluorimeter or with afluorescence microscope, or qualitatively by using the disclosedfluorescent dithio compounds as a component of a histological stain.Methods for using the fluorescent dithio compounds disclosed herein fordetecting thiol-containing compounds and/or dithio-containing compoundsare described in U.S. application Ser. No. 11/512,485, filed on Aug. 30,2006 and U.S. application Ser. No. 11/512,485, filed on Aug. 30, 2006,which are incorporated by reference herein in their entireties.

Suitable dithio compounds for the methods for detecting thiol-containingcompounds include dithio compounds having a formula F—S—S—P, in which“F” includes a fluorophore and “P” includes a non-fluorophore peptide.In the methods for detecting thiol-containing compounds anddithio-containing compounds as described herein, detecting the at leastone reaction product may include observing dequenching of thefluorophore or altered fluorescence of the fluorophore. Dequenching oraltered fluorescence may occur when the dithio compounds react withthiols that reduce the disulfide bond to form the F—S—H fluorophore (asin FIG. 1), or when the dithio compounds react with another dithio in adisulfide exchange reaction to form a new mixed disulfide.

The methods may be used to detect any suitable thiol-containing compoundand/or dithio-containing compound. Suitable compounds include any thiol-and/or dithio-containing containing compound that is capable of reducingthe fluorescent dithio compound at the dithio bond of the fluorescentdithio compound. For example, the methods may be used to detectthiol-containing compounds such as glutathione, homocysteine,cysteine-containing peptides or proteins, ADPβS, GDPβS, and combinationsthereof. Suitable dithio-containing compounds include anydithio-containing compound that is capable of reacting with thefluorescent dithio compound at the dithio bond of the fluorescent dithiocompound. Such dithios could include those in oxidized membrane-bound ormembrane-associated proteins such as the E. coli Dsb system, and itseukaryotic homologs, as well as lipid transport proteins like apoE andlipovitellin, cytokines, and C-reactive proteins (CRP). The methods fordetecting thiol-containing compounds and/or dithio-containing compoundsmay be performed in vitro, in vivo, and/or in situ. The methods may beperformed in cells. For example, the methods may be performed byadministering the fluorescent dithio compounds to tissue or cells inwhich the compounds react with at least one thiol-containing compoundand/or dithio-containing compound to form at least one reaction product.

The methods may include detecting the at least one reaction product,e.g., by fluoroscopic methods known in the art. Detecting the at leastone reaction product may include detecting dequenched fluorescence ofthe fluorophore in a reaction product using fluoroscopic methods knownin the art. Detecting the at least one reaction product may includedetecting an increase or decrease in absorbance by the fluorophore usingfluoroscopic methods known in the art. Detecting the reaction product ina reaction of the dithio compounds with thiols, will involve detectingF—S—H, and its salts. Detecting the reaction product in a reaction ofthe dithio compounds with dithios, will involve detectingF—S—S-polypeptide where the polypeptide was a dithio protein thatreacted with the original F—S—S-peptide probe. Typically, aliphaticpeptides lead to the fluorophore being quenched in the dithio compoundscontemplated herein, but polypeptides do not quench. Therefore, thedithio exchange reaction permits detection of polypeptide disulfides(i.e., F—S—S-polypeptide).

The methods for detecting thiol-containing compounds and/ordithio-containing compounds as described herein may be performedcontinuously or in real-time. As used herein, “real-time” methods aremethods in which the thiol-containing compound is detectedcontemporaneously as it is formed in a reaction mixture (e.g., as it isformed in vitro or in cells).

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and not intended to limit theclaimed subject matter.

Embodiment 1. A dithio compound having a formula F—S—S—P wherein Fcomprises a quenched fluorophore and P comprises a peptide.

Embodiment 2. The dithio compound of embodiment 1 having a formula:

Embodiment 3. The dithio compound of embodiment 1 having a formula:

Embodiment 4. The dithio compound of embodiment 1 having a formula:

Embodiment 5. The dithio compound of any of embodiments 1-4, wherein thepeptide comprises a signal peptide sequence.

Embodiment 6. The dithio compound of any of embodiments 1-5, wherein thepeptide comprises the arginine-rich RNA-binding motif (ARM) of the humanimmunodeficiency virus Tat peptide.

Embodiment 7. The dithio compound of any of embodiments 1-6, wherein thepeptide comprises an RGD sequence.

Embodiment 8. The dithio compound of any of embodiments 1-7, wherein thepeptide comprises a hormone ligand for a G-coupled cell surfacereceptor.

Embodiment 9. The dithio compound of any of embodiments 1-8, wherein thepeptide comprises an amino acid targeting sequence for a cellularnucleus.

Embodiment 10. The dithio compound of any of embodiments 1-9, whereinthe peptide comprises an amino acid targeting sequence for a cellularendoplasmic reticulum.

Embodiment 11. The dithio compound of any of embodiments 1-10, whereinthe peptide comprises an amino acid targeting sequence for a cellularmitochondria.

Embodiment 12. The dithio compound of any of embodiments 1-11 having aformula:

wherein R¹ comprises a fluorophore and R² comprises a peptide.

Embodiment 13. The dithio compound of any of embodiments 1-12, whereinthe peptide comprises no more than 20 amino acids and comprises noaromatic amino acids.

Embodiment 14. The dithio compound of any of embodiments 1-12, whereinthe peptide comprises no more than 12 amino acids and comprises noaromatic amino acids.

Embodiment 15. A method for preparing a dithio compound having a formulaF—S—S—P wherein F comprises a quenched fluorophore and P comprises apeptide, the method comprising reacting precursors that include: (A) afirst precursor that comprises a fluorophore; (B) a second precursorthat comprises a peptide; and (C) a dithio reagent having a formulaX¹—S—S—X², wherein X¹ and X² may be the same or different and eachcomprise reactive groups capable of reacting with the first precursorand the second precursor.

Embodiment 16. The method of embodiment 15, further comprising addingone or more cysteine residues to the N-terminus or C-terminus of thepeptide in order to prepare the second precursor.

Embodiment 17. A method for detecting a thiol- or dithio-containingcompound, the method comprising: (A) reacting a dithio compound having aformula F—S—S—P wherein F comprises a quenched fluorophore and Pcomprises a peptide with the thiol- or dithio-containing compound toform at least one reaction product; and (B) detecting the at least onereaction product.

Embodiment 18. The method of embodiment 17, wherein the thiol- ordithio-containing compound is a protein.

Embodiment 19. A method of preparing a dithio compound having a formulaF—S—S—P wherein F comprises a quenched fluorophore and P comprises apeptide, the method comprising reacting precursors that include: (A) afirst precursor having a formula F—S—S—F, and (B) a second precursorhaving a formula P-S—S—P.

Embodiment 20. A kit comprising: (a) a reagent having a formula F—S—S—F;and (b) a reagent having a formula P-S—S—P; wherein F is a fluorophoreand P is an aliphatic peptide.

Examples

The following examples are illustrative and not intended to limit theclaimed subject matter.

Reaction of Dithio Probe with GSH Gives a Rapid Decrease in FluorescenceFirst Before Increasing. Referring to FIG. 1, shown is a thiol reactionwith the fluorescent probe DSSA (sometimes also called DSSQ) where D isfluorescein and A is para-methyl red, a fluorescein quencher. The thiolreactant in this case is the glutathione tripeptide (GSH) at 2 mM, 4 mM,8 mM and 15 mM. DSSA probe is present at a much lower concentration, of50 uM. Reduction of any dithio probe, such as this probe (as well as aF—X¹—SS—X²-peptide), by a thiol such as the glutathione tripeptide, goesthrough a two-step process. The thiol is in excess relative to DSSA, soeventually a free fluorophore (DS) is obtained, which is morefluorescent than DSSA. The fluorescence emission spectra are shown inFIG. 1, for reaction over time at different concentrations ofglutathione. Notably, there is an initial decrease in fluorescence (dueto rapid formation of the DS-SG intermediate shown), followed by anincrease (due to subsequent formation of DS). Notably, the DSSA probeshown here still has significant fluorescence signal before reactionwith glutathione, and it goes through a two-step reaction (makingreading of fluorescence intensity more complicated). It is desirablethat a probe have a lower background fluorescence and not show thecomplication of a two-step reaction process in the fluorescence spectra.

Mixed Disulfide Intermediate Can Be Trapped by Reacting with GSSG.Referring to FIG. 2, shown is the fluorescence intensity after reactionof the probe from the previous example, but this time in a reaction withthe glutathione peptide disulfide, GSSG (rather than the reduced form ofglutathione, FIG. 1). In this case, one would expect only a disulfideexchange reaction according to the following reaction sequence:DSSQ+GSSG=>DSSG+QSSG, where D is fluorescein and GSH is glutathione. Theproduct of this disulfide exchange reaction is the same as theintermediate that was produced in FIG. 1. However, FIG. 2 demonstratesthat the DSSG intermediate, where fluorescein is tethered to theglutathione tripeptide via a disulfide bond, is even more quenched thanfluorescein in the context of the original DSSQ probe. In FIG. 3, it canbe seen that by making mixtures of reduced glutathione (GSH), withrelatively higher concentrations of the oxidized disulfide form (GSSG),there is increased level of the highly quenched intermediate (FIG. 3).This again confirms that the DSSG intermediate is highly quenched, butcan undergo further reduction (in the presence of excess GSH thiol) toform DS. This is in contrast to a pure disulfide exchange reaction, asshown in FIG. 2, where DSSG is the final product. This DSSG intermediateis one form of the F—X¹—SS—X²—P dithio compound as disclosed hereinwhere P is a peptide. This remarkable decrease in fluorescence that wasobserved upon forming the mixed disulfide with the glutathionetripeptide was unexpected. These data demonstrate that the F—X¹—SS—X²—Pdithio compound is nonfluorescent and would make a better reagent forimaging thiols and disulfides than the reagent in FIG. 1. In order totest whether some reactive nucleophilic atom on glutathione attacked thefluorescein ring, leading to loss of fluorescence, as shown in thestructure in FIG. 2, glutathione analogs were created having differentnucleophilic position chemically blocked (shown in FIG. 4). It was foundthat all of these glutathione analogs reacted with the original DSSAprobe in the same manner, with an initial decrease in fluorescence dueto formation of the F—X¹—SS—X²—P intermediate (where P is the modifiedglutathione peptide). Accordingly, quenching is occurring not because ofa nucleophilic or reactive atom on the peptide (as suggested by theproposed structure in FIG. 2), but rather because the peptide isaliphatic in nature. Subsequent studies, including quantum mechanicalcalculations, have shown that the disulfide is able to quench thefluorophore if an aliphatic peptide is present, but not if aromaticresidues or chemical groups (as in para-methyl red in FIG. 1) arepresent. Accordingly, a preferred reagent for detecting thiols anddisulfides is a fluorophore tethered, via a disulfide bond, to apeptide, because it has a lower fluorescent background signal and lesscomplicated reaction kinetics (i.e., a one step reaction).

Synthesis of dithio probes. The DSSA probe that was used in FIG. 1 wassynthesized as shown in FIG. 5. Synthesis of a DSSD probe, where twoidentical fluorophores are tethered via a disulfide bond, could beaccomplished using a slight modification of the procedure shown in FIG.5. Briefly, the first reaction to link para-methyl red and the cystaminedisulfide is omitted and excess fluorescein isothiocyanate is reacteddirectly with the cystamine disulfide.

Detection of proteins, and fluorescent properties of DSS-(protein).While previous data have demonstrated that the F—X¹—SS—X²-Peptide is notfluorescent (see FIG. 2), if proteins (more generally, polypeptides) arereacted with dithio probes, for example by reacting proteins in an SDSPAGE gel with DSSQ, they are in fact fluorescent. Therefore,F—X¹—SS—X²-Polypeptide is fluorescent, even though F—X¹—SS—X²-Peptide isnot fluorescent. For example, reaction of rabbit muscleglyceraldehyde-3-phosphate dehydrogenase in an SDS PAGE gel led to astrong fluorescent signal (FIG. 6), and reaction of the proteinsextracted from bovine lung cells, also led to fluorescent signals (FIG.7). The fact that F—X¹—SS—X²-Peptide is not fluorescent (FIG. 2), andyet can be converted to F—X¹—SS—X²-Protein via a simple disulfideexchange reaction, suggests that the F—X¹—SS—X²-Peptide dithio compoundis a suitable reagent for detecting dithio groups on proteins (but noton peptides like GSSG; see FIG. 2). This is an unexpected and highlyuseful property of fluorescent dithio reagents such asF—X¹—SS—X²-Peptide, where these reagents can be utilized in methods fordetecting dithio groups on proteins.

Confocal microscopy imaging. Fluorescent dithio probes can be used tolabel proteins in live cells. If the DSS-(protein) formed isfluorescent, then it can be visualized using confocal microscopy. Thisis shown in FIG. 8, where the chorion of zebrafish embryos was labeledusing a fluorescent dithio probe. The bright field image (FIG. 8A) showsthe fish wrapped around the yolk, and the outer layer (identified withan arrow) is the protective chorion layer. The fluorescence image inpanel B is the result of excitation at 490 nm, and shows thatfluorescence signal is coming from proteins in the chorion.Interestingly, fluorescence signal was also obtained, even if thiolswere first blocked using iodoacetamide. This suggests that labeling wasof dithio containing proteins (i.e., oxidized thiols). If so, thisrepresents the first report of imaging of dithio groups inside livecells and indicates that the dithio probes disclosed herein can beutilized in methods of labeling and imaging protein disulfides.

Disulfide proteomics. If the fluorescence signal observed in FIG. 8B isfrom disulfide containing proteins, then it should be possible to purifythem and identify them using tandem mass spectrometry (MS). In FIG. 9,when the chorion from the embryos is subjected to SDS PAGE separation,then imaged by reading fluorescence signal, it can be seen that thereare fluorescently tagged protein (DSS-protein) bands. These bands werecut from the gel and the protein contained therein was extracted andsubjected to proteolysis using trypsin. Then, peptides were subjected toLC-MS/MS analysis, where individual peptide fragments are ionized in amass spectrometer (ESI, in this case), and the mass/charge fragmentationpattern matched against a database of predicted patterns for theproteome in question (in this case, zebrafish). Using this approach,patterns were obtained for the two bands from the gel in FIG. 9 (Bands Land H) (data not shown). In this manner, Band L (21,097g/mol) wasidentified as being from the zebrafish homolog of lipovitellin, a lipidbinding protein. Likewise, Band H (23,675 g/mol) was identified as beingfrom the zebrafish homolog of C-reactive protein (CRP), which is animportant biomarker protein in humans associated with infection,inflammation and tissue damage. When these proteins were subjected tohomology modeling analysis, using the Swiss Model Server (data notshown), high quality homology models could be obtained due to highsequence identity with the template proteins (>30% identity). The3-dimensional protein structure models clearly indicate that bothproteins have disulfide groups, consistent with the above conclusionthat the fluorescent dithio probes were reacting with disulfide groupson proteins in the chorion. Accordingly, fluorescent dithio probes canbe used in dithio proteomics methods. Furthermore, given the broadclinical important of C-reactive protein, and in general of detectingtissue damage, the fluorescent dithio probes may have utility as aclinical diagnostic reagent to identify tissue damage, infection orinflammation. In this regard, it has been noted that C-reactive proteinand lipid transport proteins, in humans, may play a role in lipidtransport and tissue damage.

Summary. The results illustrate that there is utility in tetheringfluorescein or other fluorophores to aliphatic systems like peptides(e.g., fluorescein-S—S-peptide).

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

1. A dithio compound having a formula F—S—S—P wherein F comprises aquenched fluorophore and P comprises a peptide.
 2. The dithio compoundof claim 1 having a formula:


3. The dithio compound of claim 1 having a formula:


4. The dithio compound of claim 1 having a formula:


5. The dithio compound of claim 1, wherein the peptide comprises asignal peptide sequence.
 6. The dithio compound of claim 1, wherein thepeptide comprises the arginine-rich RNA-binding motif (ARM) of the humanimmunodeficiency virus Tat peptide.
 7. The dithio compound of claim 1,wherein the peptide comprises an RGD sequence.
 8. The dithio compound ofclaim, wherein the peptide comprises a hormone ligand for a G-coupledcell surface receptor.
 9. The dithio compound of claim 1, wherein thepeptide comprises an amino acid targeting sequence for a cellularnucleus.
 10. The dithio compound of claim 1, wherein the peptidecomprises an amino acid targeting sequence for a cellular endoplasmicreticulum.
 11. The dithio compound of claim 1, wherein the peptidecomprises an amino acid targeting sequence for a cellular mitochondria.12. The dithio compound of claim 1 having a formula F—X¹—S—S—X²—P,wherein at least one of X¹ and X² comprises an aryl group.
 13. Thedithio compound of claim 1 having a formula:

wherein R¹ comprises a fluorophore and R² comprises a peptide.
 14. Thedithio compound of claim 1 having a formula:

wherein R¹ comprises a fluorophore and R² comprises a peptide.
 15. Amethod for preparing a dithio compound having a formula F—S—S—P whereinF comprises a quenched fluorophore and P comprises a peptide, the methodcomprising reacting precursors that include: (A) a first precursor thatcomprises a fluorophore; (B) a second precursor that comprises apeptide; and (C) a dithio reagent having a formula X¹—S—S—X², wherein X¹and X² may be the same or different and each comprise reactive groupscapable of reacting with the first precursor and the second precursor.16. The method of claim 15, further comprising adding one or morecysteine residues to the N-terminus or C-terminus of the peptide inorder to prepare the second precursor.
 17. A method for detecting athiol- or dithio-containing compound, the method comprising: (A)reacting a dithio compound having a formula F—S—S—P wherein F comprisesa quenched fluorophore and P comprises a peptide with the thiol- ordithio-containing compound to form at least one reaction product; and(B) detecting the at least one reaction product.
 18. The method of claim17, wherein the thiol- or dithio-containing compound is a protein.
 19. Amethod of preparing a dithio compound having a formula F—S—S—P wherein Fcomprises a quenched fluorophore and P comprises a peptide, the methodcomprising reacting precursors that include: (A) a first precursorhaving a formula F—S—S—F, and (B) a second precursor having a formulaP-S—S—P.
 20. A kit comprising: (a) a reagent having a formula F—S—S—F;and (b) a reagent having a formula P-S—S—P; wherein F is a fluorophoreand P is an aliphatic peptide.